Abstract
Amyotrophic Lateral Sclerosis, or ALS, is the most common neuromuscular disorder, affecting approximately 5600 Americans each year. It is caused by the degeneration of both upper and lower motor neurons, and those diagnosed only have 2-3 years before death due to progressive muscle paralysis and respiratory failure. The underlying mechanism of the disease is unknown, though it is theorized to be a mixture of cellular processes such as genetics, oxidative stress and excitotoxicity, where nerve cells are damaged by excessive stimulation by neurotransmitters. There is no standardized form of diagnosis and no biomarkers for ALS. Discovery of biomarkers would help to properly diagnose those with ALS, as well as accelerate drug development. One method of finding a biomarker is through mass spectrometry, more specifically MALDI-MS imaging. MALDI-MS imaging creates a map of ion intensities across a given section of tissue based on protein concentration and spatial distribution. In this study, we optimize a set of parameters to use in the preparation of samples for MALDI-MS imaging, since there is no universal method yet developed. Optimization was performed with META (8:8:1:1 methanol, ethanol, acetonitrile and 0.1% TFA in water), a matrix solvent that fixes proteins in their respective locations in the tissue and found an optimum exists at 3 depositions at 20 mg/mL. We also discovered that the number of depositions of a given concentration of matrix solution makes a very large difference in the quality of spectra obtained, even more so than the total concentration of matrix on the tissue. This discovery was significant, and we then applied our discovery to a more general MALDI solvent – a 50/50 mixture of acetonitrile and 1% trifluoracetic acid (TFA) in water. We tested the same parameters – number of depositions and initial concentration to find that they also play a large role in the spectral quality obtained with this matrix solvent. The optimal conditions for this solvent existed at 5 depositions of 20 mg/mL. We also discovered that in addition to an effective matrix solvent, META is an excellent wash solvent. Use of a META wash before matrix application resulted in more sensitive and reproducible spectra, with higher intensity and lower variability and preserved fluorescence of transgenic YFP tissue. Optimal conditions were applied to a comparison study with G93A positive and control tissue in the motor cortex, and preliminary differences in spectra acquired were discovered. Future experiments will explore these differences further, both in the motor cortex and spinal cord to facilitate the discovery of a biomarker for ALS.